Flexible Fitting of Atomic Models in cryoEM Density (Flex ...
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Maya Topf
Flexible Fitting of Atomic Models in cryoEM Density
(Flex-EM, RIBFIND & TEMPy)
S2C2 CryoEM CCP-EM Modeling Workshop11th Nov 2020
EMDB Statistics
https://www.ebi.ac.uk/pdbe/emdb/statistics_main.html/
Villa & Lasker, Curr Opin Struct Biol, 2014
Structural features vs. resolution
De novo
Flexible Fitting
Rigid-Body Fitting / Assembly Fitting
25 Å
Aims:
• To understand how to optimise your structural model inside the 3D-EM density map, with focus on Flex-EM and RIBFIND
• To understand approaches to model validation in the context of 3D-EM maps, with focus on TEMPy
Approach to modelling structures in 3D-EM maps
Multi-componentrigidfitting
Fitassessment
Map+Model
Flexiblefitting(refinement)
TEMPy
Flex-EM/RIBFINDγ-TEMPy
tempy.ismb.lon.ac.uktopf-group.ismb.lon.ac.uk/flex-em
• Uses simulated annealing rigid-body dynamics.
• During the refinement, the atoms (or rigid bodies) are displaced in the direction that maximizes their cross-correlation with the cryoEM density map (ECCC) and minimizes the violations of the stereochemical (ESC) and non-bonded contacts (ENB).
• Restraints are used to reduce over-fitting and maintain geometry.
• Rigid body restraints are defined based on inter-atomic or inter-residue contacts. Atoms in a rigid body move together during the course of refinement.
Topf et al. Structure 2008 http://topf-group.ismb.lon.ac.uk/flex-em/
Flex-EM: flexible fitting by real-space refinement
salilab.org/modeller
• Flex-EM can use rigid bodies calculated with RIBFIND.
• RIBFIND works by clustering secondary structure elements together (and intermittent regions) as rigid bodies.
Pandurangan et al. JSB 2012
Rigid-body restraints
SSE-based clustering
• Allows faster large body movements in the initial stages of refinement
• Useful when the resolution of density map is insufficient to fit smaller entities like individual residues or atoms.
0%
Cluster cutoff100%25% 35% 37% 50% 51%
Numberofclusters1 2 3 2 0
https://ribfind2.ismb.lon.ac.uk/
RIBFIND
PDB: 2driA 10Å resolution map
Pandurangan et al. JSB 2012
Initial Un-clustered Clustered
Cα RMSD from X-ray structure:
Hierarchical refinement
PDB: 1dpe 5Å resolution map Initial Final un-clustered Final clustered
Cα RMSD: 12.28Å 3.92Å3.69Å un-cluster0.81 0.870.89CCC:
Final two-stage refinement
RMSD:2.12ÅCCC:0.92
Pandurangan et al. JSB 2012; Clare et al., Cell 2012
Joseph et al. Methods 2016
Hierarchical fitting with Flex-EM
GroEL at 4.1 Å (EMD: 6422)
Flex-EM approach to high resolutions
• For large body motions, the maximum atom displacement along one axis at each MD step is limited by 0.39 Å.
• In cases where the model does not have multiple domains/sub-domains that can be identified as large RBs, one can start with the stage where SSEs are constrained as rigid. At this stage, maximum atom shifts were limited to ~0.2 Å at each MD step.
• At the final refinement stage, relative motions between all atoms were allowed without considering any rigid segments. The maximum atom displacement steps are reduced to 0.1 Å in this final stage for finer fitting refinement.
Implementation in CCP-EM
RIBFIND Flex-EM
Model assessment with TEMPy
http://tempy.ismb.lon.ac.uk/
Cragnolini et al. TEMPy2: A python library with improved 3D electron microscopy density fitting and validation workflows. Acta Crystallogr. D (in press).
http://tempy.ismb.lon.ac.uk/
Cragnolini et al. TEMPy2: A python library with improved 3D electron microscopy density fitting and validation workflows. Acta Crystallogr. D (in press).
TEMPy: Template and EM comparison using Python
Multiple TEMPy scores to compare model fits globally
Implementation in CCP-EM
Joseph et al. JSB 2017
• Cross-correlation coefficient (CCC)
• Mutual information-based score (MI)
• Model-map overlap
Local scoring
TEMPy + Chimera attribute files
Useful for calculating CCC locally on any defined segment
Probe density X
Target density Y
SCCC = SCCC
Segment-based CCC:
Pandurangan et al., 2014; Farabella et al. 2015; Atherton et al. eLIFE (2017)
Local scoring
Segment-based manders’ overlap coefficient:
TEMPy + Chimera
Useful to calculate local fit per residue
Joseph AP. et al. Methods 2016
SM
OC
The overlap coefficient is calculated over voxels covered by each residue (and the local neighbourhood), both individually (SMOCd) and along the chain (SMOCf)
Local scoring
Segment-based manders’ overlap coefficient:
Joseph AP. et al. Methods 2016
The overlap coefficient is calculated over voxels covered by each residue (and the local neighbourhood)
EMD-3488 (3.2Å) Deposited model PDB: 5NI1
329_1o: best by CCC (TEMPy) 460_1o: best by lDDT
Target contains errors
TS0984o
Example from CASP13
https://predictioncenter.org/casp13/Kryshtafovych, Malhotra et al. Proteins 2019
Joseph A.P. et al. Methods 2016
Unliganded GroEL at 4.2 Å resolution - (EMD-5001)ADP-bound GroEL (PDB: 4KI8)Refined modelDeposited model (Ludtke et al. Structure 2008, PDB: 3cau)
Hierarchical Refinement at intermediate to near-atomic resolutions
Implementation in CCP-EM
Joseph et al. JCIM 2020, Locke et al. PNAS 2017
Calculating difference maps
Agnel Joseph
Irene Falabella Daven Vasishtan
Arun P Pandurangan
Approach to modelling structures from 3D-EM maps
Harpal Sahota
Sony Malhotra
Tristan Cragnolini
Multi-componentrigidfitting
Fitassessment
Map+Model
Flexiblefitting(refinement)
TEMPy
Flex-EM/RIBFINDγ-TEMPy
Ben Blundell
Thomas Mulvaney
Aaron Sweeney
Thanks…
Topf group (current)Sony Malhotra Tristan Cragnolini Mauro Maiorca Aaron Sweeney Rebecca Brooker Eric Escriva Guendalina Marini Sophie Knott Tomas Mulvaney Victoria Sanders Manaz Kaleel Karen Manalastas
CCP-EM (STFC)Martyn Winn Tom Burnley Colin Palmer Agnel-Praveen Joseph
EM groupNatasha Lukoyanova Helen Saibil Carolyn Moores Joe Atherton Alex Cook Elena Orlova
ISMB UCL/BirkbeckDave Houldershaw Kostas Thalassinos
EMDB (EBI)Ardan Patwardhan Gerard Kleywegt Ingvar Lagerstedt
eBICDan Clare
HPI (Hamburg)/OxfordKay Grünewald Daven Vasishtan
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